Chemical Permeation Enhancers Enhance Nerve Blockade by Toxins

ABSTRACT

Chemical permeation enhancers (CPEs) improve access of local anesthetics to the nerve, thereby improving their performance. Surfactants, representing three CPE sub-groups: anionic, cationic, and nonionic surfactants, were co-injected with tetrodotoxin (TTX) or bupivacaine at the sciatic nerve of Sprague-Dawley rats. All enhancers produced marked concentration-dependent improvements in the frequency and duration of block with TTX but not bupivacaine. An in vitro toxicity assay showed a wide range of CPE myotoxicity, but in vivo histological assessment showed no signs of muscle or nerve damage at concentrations of CPEs that produced a half-maximal increase in the duration of block of TTX. There was no systematic relationship between the enhancers&#39; charge or hydrophobicity and their enhancement of block duration or potency. Thus, CPEs can provide marked prolongation of nerve blockade from TTX, without apparent local tissue toxicity, and therefore enhance the clinical applicability of TTX for prolonged-duration local anesthesia.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. GM073626awarded by National Institute of General Medical Sciences. Thegovernment has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application No. 61/954398, fifed on May 19, 2008, entitled“Chemical Permeation Enhancers of Nerve Blockade” by Daniel S. Kohaneand Emmanuel J. Simons, and where permissible is incorporated byreference in its entirety.

FIELD Of THE INVENTION

This relates generally to methods and compositions enhancing nerveblockade with local, anesthetics.

BACKGROUND OF THE INVENTION

Local anesthetics must penetrate the epineurium, perineurium, andendoneurium in order to reach their intended sight of action.Consequently, local anesthetics require ranch higher concentrations tobe effective when used clinically than in isolated nerves (Schwartz, etah, J Physiol 233:167-494 (1973); Hahin, et. al;, J Gen. Physiol,78:113-139 (1981); Chernof, et al., Biophys J., 58:69-81 (1990); Lee, etal., Anesthesiology, 77:324-335 (1992); Kohane, et al, Anesthesiology,89:1199-1208 (1998)). The literature suggests that a small molecule'shydrophobicity has a U-shaped, effect on its ability to penetratebiological barriers (Bernards and H.F. Hill, Anesthesiology 77(4):750-6(1992)): drugs with an intermediate degree of hydrophobicity penetratemore effectively than those that are very hydrophobic or veryhydrophilic. There are some data to suggest that this relationship holdstrue for local anesthetics penetrating to or into peripheral nerve(Barnet et al, Pain 110:432-438 (2004)).

A number of methods have been used in the attempt to increase theduration of action of local anesthetics. A method currently used inmedical practice is the co-administration of vasoconstrictors such asepinephrine (adrenaline), phenylephrine, or norepinephrine, whichincrease the residence time of the drug at the site of administration,due to the induction of vasoconstriction with subsequent reduction ofsystems uptake of the local anesthetic or biodegradable polymer matrices(U.S. Pat. No. 5,618,563) and glucocorticoids (U.S. Pat. No. 5,700,485).

Chemical permeation enhancers (CPEs) have been used to increase thepermeability of the lipid-protein barriers of the skin, and therebyincrease drug flux, for over thirty years (Banerova, K. et at, Eur JDrug Metab Pharmacokinet 200126(1-2): 85-94; Asbill, C.S., et al. CritRev Ther Drug Carrier Syst 17(6); 621-658 (2000) Kanikkannan, N., CurrMed Chem 7(6):593-608 (2000); Karande, et al, J Control Release110:307-313 (2006)). Surfactants, a heterogeneous group of amphophilicorganic molecules with hydrophilic heads and hydrophobic tails, are awell-known class of CPEs. Several sub-classes of surfactants have beenstudied in the context of transdermal permeation, and are believed toreversibly modify lipids by adsorption at interfaces and removal ofwater-soluble agents that act as plasticizers (Middleton, J.D. J SocCosmet Chem 20:399-403 (1969); Ribaud, C., et al. Pharm Res 11:1414-1418(1994)). Cationic surfactants are known to produce greater increases inpermeant flux than anionic surfactants, which, in turn, increasepermeability more than nonionic surfactants (Stoughton, R.B. In: E.M.Farber (Ed.), Psoriasis, Grune and Stratton, Orlando, Fla., 1982, p.346-398; Kushla. et al., J Pharm Sci 82:1118-1122 (1993); Shen, et al.,J Pharm Sci 65:1780-1783 (1976)). A broad range of non-surfactantchemical enhancers has also been investigated (e.g., alcohols,sulfoxides, polyols, fatty acids, esters, terpenes, and cyclodextrins),(Middleton, J Soc Cosmet Chem 20:399-403 (1969); Riband, C., et al.,Pharm Res 11:1414-1418 (1994): Stoughton, R.B., In: E.M. Farber (Ed.),Psoriasis, Grune and Station, Orlando, Fla., 1982, p. 346-398; Kushla,G.P., J Pharm Sci, 82:1118-1122 (1993); Shen, W. W., et a., J Pharm Sci65:1780-1783 (1976); R. B. Walker and E. W. Smith, Adv Drug DeliveryRev, 18:295-301 (1996)).

U.S. Pat. No. 6,455,066 to Fischer, et al., for example, describes theuse of intradermal penetrating agents triglyceride, aloe composition,and a mixture thereof for topical local anesthetic administration. U.S.Pat. No. 6,673,363 to Luo, et al. describes the use of organic orinorganic permeation enhancers for the delivery of anesthetic agents.U.S. Pat. No. 6,326,020 to Kohane, et al., describes the combination ofnaturally occurring site I sodium channel blockers such as tetrodotoxinwith other agents such as another local anesthetic, a vasoconstrictor,glucocorticoid, adrenergic drugs, or amphophilic or lipophilic solventto improve the duration of block.

There is still a need for a composition that can provide prolonged nerveblock while avoiding systemic toxicity.

It is an object of the present invention to provide a composition foruse as an anesthetic with increased potency and efficacy.

It is still another method of the invention to provide a method forlocal anesthesia that avoids systemic toxicity due to the localanesthetic and provides prolonged nerve block.

SUMMARY OF THE INVENTION

Combinations of site I sodium channel blocker local anesthetics withchemical penetration enhancers have been developed to improve thepotency and efficacy of local anesthetics, thereby decreasing theirsystemic toxicity without increasing local toxicity. The duration ofblock is greatly prolonged by combining the local, anesthetic with achemical penetration enhancer. In one embodiment, the local anestheticis a hydrophilic local anesthetic. In a preferred embodiment, the local,anesthetic is a Site I sodium channel blocker such as tetrodotoxin.Chemical permeation enhancers (CPEs) improve access of local anestheticsto the nerve, thereby improving their performance.

Surfactants, representing three CPE sob-groups; anionic, cationic, andnonionic surfactants, were co-injected with tetrodotoxin (TTX) orbupivacaine at the sciatic nerve of Sprague-Dawley rats. All enhancersproduced marked concentration-dependent improvements in the frequencyand duration of block with TTX but not bupivacaine. An in vitro toxicityassay showed a wide range of CPE myotoxicity, but in vivo histologicalassessment showed no signs of muscle or nerve damage at concentrationsof CPEs that produced a half-maximal increase is the duration of blockof TTX. There was no systematic relationship between the enhancers'charge or hydrophobicity and their enhancement of block duration orpotency. Thus, CPEs can provide marked prolongation of nerve blockadefrom TTX, without apparent local tissue toxicity, and therefore enhancethe clinical applicability of TTX for prolonged-duration localanesthesia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of different chemical penetration.enhancers (SOS, SLS, OTAB, DDAB, TW 20, and TW 80) on the duration ofsensory block from 30 μM TTX.

FIG. 2 is shows the survival of C2C12 myotubes after a 2-hour exposureto PBS or TTX alone (control) or to the EC_(50eff) (CPE concentrationthat caused a half-maximal increase in block duration) of each CPE (SOS,SLS, OTAB, DDAB, Tween® 20 and Tween® 80) alone and with 30 μM TTX. Dataare shown as means ± standard deviations (n=4).

FIG. 3 is a graph showing the maximum block duration (MBD) plottedagainst interpolated EC_(100min) values for each CPE. Anionic, cationicand nonionic surfactants are grouped by color (black, grey, and whiterespectively). Block durations are expressed as medians with 25^(th) and75^(th) percentiles (n=4).

FIG. 4 shows MBD and EC_(100min) plotted against cell survival. Cellsurvival data are means with standard deviations from C2C12 cellsexposed to each CPE at its EC_(50eff) for two hours. Block durations areexpressed as medians with 25^(th) and 75^(th) percentiles.

FIG. 5A is a graph of the effect of 50 μM TTX on the duration ofeffective motor (light grey) and thermal nociceptive (dark) block(minutes) from 100 mM QX-222 and 25 mM QX-314.

FIG. 5B is a graph of the effect of 30 and 40 μM TTX on the duration ofeffective motor (light grey) and thermal nociceptive (white) block(minutes) from 25 and 70 mM QX-314

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, chemical penetration enhancer (CPE) denotes any agentwhich can alter a biological barrier to enhance permeant flux or achemical agent capable of reducing the surface tension of a liquid inwhich it is dissolved. The CPE is preferentially a surfactant, mostpreferably a cationic surfactant.

As used herein, local anesthetic (LA) refers to any agent that producesnerve blockage within a specific area, region or site.

“Aryl”, as used herein, refers to -, 6- and 7-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring system, optionally substituted by halogens, alkyl-,alkenyl-, and alkynyl- groups. Broadly defined, “Ar”, as used herein,includes 5-, 6- and 7-membered single-ring aromatic groups that mayinclude from zero to four heteroatoms, for example, benzene, pyrrole,furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,pyridine, pyrazine, pyidazine and pyrrimidine, and the like. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles” or “heteroaromatics”. The aromatic ring can besubstituted at one or more ring positions with such substituents asdescribed above, for example, halogen, azide, alkyl aralkyl, alkenyl,alkynyl cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,amido, phosphonate, phosphinate, carbonyl carboxyl, silyl, ether,alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl,aromatic or heteroaromatic moieties, —CF3, —CN, or the like. The term“Ar” also includes polycyclic ring systems having two or more cyclicrings in which two or more carbons are common to two adjoining rings(the rings are “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic-ring include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H, 6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxaxolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

“Alkyl”, as used herein, refers to the radical of saturated orunsaturated aliphatic groups, including straight-chain alkyl, alkenyl,or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups,cycloalkyl, cycloalkenyl, or cycloalkynyl (alicycllc) groups, alkylsubstituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, andcycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unlessotherwise indicated, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C1-C30 for straight chain,C3-C30) for branched chain), preferably 20 or fewer carbons, morepreferably 10 or fewer carbons, most preferably 5 or fewer carbons.Likewise, preferred cycloalkyls have from 3-10 carbon atoms in theirring structure, and more preferably have 5, 6 or 7 carbons in the ringstructure. The alkyl group can be substituted with one or moresubstituents including, but not limited to, alkyl, aryl, halogen,hydroxy, and thiol.

II. Compositions

The composition is designed to prolong the duration of a localanesthetic block, with no systemic toxicity. The composition consist ofa local anesthetic in combination with one or more chemical penetrationenhancers in amounts effective to prolong the duration of block of thelocal, anesthetic, with no significant systemic toxicity, and isadministered in a pharmaceutically acceptable carrier. The preferredcomposition contains a site I sodium channel blocker. The composition isadministered in a formulation locally at the site where the nerve is tobe blocked, preferably as a solution. The chemical penetration enhanceris preferably a charged penetration enhancer. In a preferred embodiment,the chemical penetration enhancer is a charged surfactant,

A. Site I Sodium Channel Blocker Local Anesthetics

Site I sodiom channel blockers include tetrodotoxin (TTX), saxitoxin(STX), decarbamoyl saxitoxin, neosaxitoxin, and the gonyautoxins(referred to jointly herein as “toxins”). The preferred toxins are TTXand SIX.

Tetrodotoxins are obtained from the ovaries and eggs of several speciesof puffer fish and certain species of California newts. Chemically, itis an amino perhydroquinaoline. See Kao, Pharmacological Reviews,18(2):997-1049 (1966). Tetrodofoxm. alone is too toxic to be used as ananesthetic.

Saxitoxm was first extracted from the Alaska butterclam, Saxidomusgigantuus, where it is present in algae of the genus Gonyaulax. Thereported chemical formula is C₁₀H₁₅N₇O₃.2HCl. It is believed the toxinhas a perhydropurine nucleus in which are incorporated two guanidiniummoieties. Saxitoxin is too toxic to be used alone as a local anesthetic.

A number of unusual polypeptides have been isolated from the paralyticvenoms of the fish hunting cone snails of the genns Conus found in thePhilippine archipelago. Many of these, designated “conotoxins,” havebeen discovered to affect ion channel function. The paralytic a, m, andw conotoxins block nicotinic acetylcholine receptors, sodium channels,and voltage sensitive calcium channels, respectively (reviewed inOlivera, et. al., 249:257-263 (1990)). Those which block sodium channelscan be used in the same manner as the tetrodotoxins and saxitoxins.

B. Chemical Penetration Enhancers

Any biocompatible CPE can be administered in combination with the localanesthetic, either slightly before, after or with the local anesthetic.

Suitable penetration enhancers include sulfoxide decylmethylsulfoxide(C₁₀MSO); ethers snob as diethylene glycol monoethyl ether,dekaoxyethylene-oleylether, and diethylene glycol monomethyl ethers;surfactants such as sodium lauryl sulfate (SLS), sodium octyl salfate(SOS), dodecyltriethylammonium bromide (DDAB), octyltriethylammoniumbromide (OTAB), TWEEN® 20 and TWEEN® 80, fatty acids such as C8-C22 andother fatty acids, C8-C22 fatty alcohols, and polyols.

Surfactants can be used in combination with other (non-surfactant) CPEsto enhance blockade.

The preferred CPEs are shown in Table 1:

TABLE 1 Properties of the chemical penetration enhancers (CPEs). CPE MWLength of Carbon Chain Sodium Ocryl Sulfate (SOS) 232.28 8 SodiumDodecyl Sulfate (SLS) 288.38 12 Octyl-trimethyl-ammonium 252.23 8Bromide (OTAB) Dodecyl-trimethyl-ammonium 308.34 12 Bromide (DDAB) Tween20 (TW 20) 1228 12 Tween 80 (TW 80) 1310 17

These have the following chemical structures.

CPE/ CLASS Structure SOS Anionic

SLS Anionic

OTAB Cationic

DDAB Cationic

TW 20 Nonionic

TW 80 Nonionic

Other suitable penetration enhancers include, but aie not limited to,urea, (carbonyldiamide), imidurea, N, N-diethylformamide,N-methyl-2-pyrrolidine, 1-dodecal-azacyclopheptane-2-one, calciumthiogilycate, 2-pyyrolidine, N,N-diethyl-m-toluamide, oleic acid and itsester derivatives, such as methyl, ethyl, propyl, isopropyl, butyl,vinyl and glycerylmonooleate, sorbitan esters, such us sorbitanmonolaurate and sorbitan monooleate, other fatty acid esters such asisopropyl laurate, isopropyl myristate, isopropyl palmitate,diisopropyl] adipate, propylene glycol monolaurate, propylene glycolmonooleatea and non-ionic detergents such as Brij® 76 (stearyl poly(10oxyethylene ether), Brij® 78 (stearyl poly(20)oxyethylene ether). Brij®96 (oleyl poly(10)oxyethylene ether), and Brij® 721 (stearyl poly (21)oxyethylene ether) (ICI Americas Inc, Corp.). Fatty acids such aslinoleic acid, capric acid, lauric acid, and neodecanoic acid, which canbe in a solvent such as ethanol or propylene glycol, can be used aslipid bilayer disrupting agents. Vegetable oils, such as peanut oil, mayalso be used as a penetration enhancer.

U.S. Pat. No. 4,537,776 to Cooper contains a summary of prior art andbackground information detailing the use of certain binary systems forpermeant enhancement. European Patent Application 43,738, also describesthe use of selected diols as solvents along with a broad category ofcell-envelope disordering compounds for delivery of lipophilicpharmacologically-active compounds. A binary system for enhancingmetaclopramide penetration is disclosed in UK Patent Application GB2,153,223 A, consisting of a monovalent alcohol ester of a C8-32aliphatic monocarboxylic acid (unsaturated and/or branched if C18-32) ora C6-24 aliphatic monoalcohol (unsaturated and/or branched if C14-24)and an N-cyclic compound such as 2-pyrrolidone or N-methylpyrrolidone.

Combinations of enhancers consisting of diethylene glycol monoethyl ormonoethyl ether with propylene glycol monolaurate and methyl laurate aredisclosed in U.S. Pat. No. 4,973,468 for enhancing the transdermaldelivery of steroids such as progestogens and estrogens. A dual enhancerconsisting of glycerol monolaurate and ethanol for the transdermaldelivery of drugs is described in U.S. Pat. No. 4,820,720, U.S. Pat. No.5,006,342 lists numerous enhancers for transdermal drug administrationconsisting of fatty acid esters or fatty alcohol ethers of C₂ to C₄alkanediols, where each fatty acid/alcohol portion of the ester/ether isof abont 8 to 22 carbon atoms. U.S. Pat. No. 4,863,970 disclosespenetration-enhancing compositions for topical application including anactive permeant contained in a penetration-enhancing vehicle containingspecified amounts of one or more cell-envelope disordering compoundssuch as oleic acid, oleyl alcohol, and glycerol esters of oleic acid; aC₂ or C₃ alkanol and an inert diluent such as water.

Liposomes are microscopic aggregates if highly ordered lipid moleculeswhich are normally dispersed in a hydrophilic solvent. Liposomes havebeen shown to enhance the permeability of drugs (reviewed in Choi, eta.l., J. Pharmacol and Biophys. Res., 18(5):209-19 (2005). In anotheremhodiment, suspensions in chromophobes conventionally used in the artto enhance permeation are used. The local anesthetic can also beadministered as an emulsion, such as an oil-in-water or a water-in-oilemulsion.

C. Combinations of Anesthetic and Active Agents and/or CPE

The local anesthetic and CPE can be combined into a single dosage formor sequentially administered. The effective amount and ratio of CPE toanesthetic is dependent on the anesthetic, the CPE, the site ofadministration, and the species into which the anesthetic isadministered. More specifically, dosage and concentrations will changedepending on the size of nerve, species, anatomic location (peripheralnerve, epidural space, intrathecal), and even the volume of injestate.The concentration and dosages can be determined as demonstrated in theexamples.

In general, the concentrations will be within the following ranges,although the range may be greater.

Site I sodium channel blockers:For TXX: 10-120 micromolar,For saxitoxin: 5-60 microloarFor neosaxitoxin: 3-40 micromolarFor decarbamoyl STX 30-480 micromolar

These numbers are derived from: Kohane DS, Lu NT, Gokgol-Kline AC,Shubina M, Kuang Y, Hall S, Stricter GR, Berde CB, “The localanesthietic properties and toxicity of saxitonin homologies for ratsciatic nerve block in vivo”, Reg Anesth Pain Med 25: 52-9 (2000).

In applications where the volume delivered is very small, theconcentrations could be up to 100 times higher.

CPE range of concentrations:

SOS: 10-100 mM SLS: 1-40-mM DDAB: 0.5-10 nM OTAB: 20-120 mM Tween®20:3-50 mM Tween® 80:20-80 mM

These numbers are derived from: Simons EI, Bellas E, Lawlor MW, KohaneDS: Effect of chemical permeation enhancers on nerve blockade, MolPharmaceutics 2009; 6: 265-273.

In another embodiment these agents are co-injected with avasoconstrictor.

In still another embodiment the site I sodium channel blocker iscombined with another local anesthetic. Useful local anesthetics includeamino-amide or amino-ester local anesthetics, any at least partlyamphiphilic local anesthetics, local anesthetics that act not on thesurface of the cell, and any at least partly charged local anesthetics.

In one embodiment, the local anesthetic is a charged local anesthetic,preferably a permanently charged local anesthetic. Preferred chargedlocal, anesthetics are those of Formnla I or Formula II:

where R₁-R₅ are independently selected from hydrogen; linear, branched,or cyclic alkyl and aryl groups.

Suitable local charged anesthetics of Formula I and II include, but arenot limited to, charged lidocaine derivatives, such asQX-314((N-(2,6)dimethylphenylcarbarmoylmethyl triethylammonium bromide);QX-222 (2-((2,6-dimethylphenyl)amino-N,N,N-trimethyl-2-oxoethanaminium); QX-572(N,N-bis(phenylcarbomoylmethyl)-dimethylammonium chloride).

QX-314 is a quaternary lidocaine derivative that is permanently chargedand lipophobic. QX-314 is a powerful blocker of voltage-sensitive Na+conductance when applied intracellularly. QX-314 suppresses thegeneration of Na+-dependent spikes from inside the cell membrane,without affecting Ca2+ currents or glutamate-activated currents. Othersuitable charged anesthetics include, but are not limited to, tonicaine.

The structures of QX-314, QX-222, QX-572, and tonicaine are shown below:

Other suitable charged local anesthetics include, but are not limitedto, charged tetracaine derivatives (e.g., N-butyl tetracaine) andpermanently charged derivatives of flecainide.

In one embodiment, the local anesthetic is in an excipient having a pHthat causes the local anesthetic to be charged.

D. Formulations

The compounds described herein can be formulated for parenteral ortopical formulation. The compounds can be combined with one or morepharmaceutically acceptable carriers and/or excipients that areconsidered safe and effective and may be administered to an individualwithout causing undesirable biological side effects or unwantedinteractions. The carrier is all components present im thepharmaceutical formulation other than the active ingredient oringredients.

Parenteral Formulations

The compounds described herein can be formulated for parenteraladministration. “Parenteral administration”, as used herein, meansadministration by injection.

The preparation of an aqueous composition that contains one or more ofthe compounds described herein is known in the art. Typically, suchcompositions can be prepared as injectable formulations, for example,solutions or suspensions; solid forms suitable for using to preparesolutions or suspensions upon the addition of a reconstiration mediumprior to injection; emulsions, such as water-in-oil (w/o) emulsions,oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, andemulsomes (see U.S. Pat. No. 5,716,637 to Anselem et al.).

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Solutions of the active compounds as the free acid or base orpharmacologically acceptable salts thereof can be prepared in watersuitably mixed with one or more pharmaceutically acceptable excipientsincluding, but not limited to, surfactants, dispersants, emulsifiers, pHmodifying agents, and combination thereof. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils, such as vegetable oils, e.g., peanut oil, corn oil, sesameoil, etc. Dispersions can contain one or more of the pharmacentleallyacceptable excipients listed above.

Suitable surfactants to facilitate formulation may be anionic, cationic,amphoteric or nonionic surface active agents. Suitable anionicsurfactants include, but are not limbed to, those containingcarboxylate, sulfonate and sulfate ions. Examples of anionic surfactantsinclude sodium, potassium, ammonium of long chain alkyl sulfonates andalkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkylsodium, sulfosuccinates, such as sodium dodecylbenzene sulfonate;dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401 ,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfectants include sodiumN-dodecyl-beta-alanine, sodium. N-lauryl-.beta.-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

Under ordinary conditions of storage and use, the formulation cancontain a preservative to prevent the growth of microorganisms. Suitablepreservatives include, but are not limited to, parabens, chlorobutanol,phenol, sorble acid, and thimerosal. The formulation may also contain anantioxidant to prevent degradation of the active agent(s).

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffets, and citrate buffers.

Water soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carbox ymethylcellulose, andpolyethylene glycol.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent ordispersion medium with one or more of the excipients listed above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those listed above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The powders can be prepared in such a manner that theparticles are porous in nature, which can increase dissolution of theparticles. Methods for making porous particles are well known in theart.

Controlled release formulations

The parenteral formulations described herein can be formulated forcontrolled release including immediate release, delayed release,extended release, pulsatile release, and combinations thereof. Thecompositions can be incorporated into microparticles, nanoparticles, orcombinations thereof that provide controlled release. In embodimentswherein the formulations contains two or more drugs, the drugs can beformulated for the same type of controlled release (e.g., delayed,extended, immediate, or pulsatile) or the drugs can be independentlyformulated for different types of release (e.g., immediate and delayed,immediate and extended, delayed and extended, delayed and pulsatile,etc.).

Release of the drug(s) is controlled by diffusion of the drug(s) out ofthe microparticles and/or degradation of the polymeric particles byhydrolysis and/of enzymatic degradation. Suitable polymers includeethylcellulose and other natural or synthetic cellulose derivatives.Polymers which are slowly soluble and form a gel in an aqueousenvironment, such as hydroxypropyl methylcellulose or polyethylene oxidemay also be suitable as materials for drug containing microparticles.Other polymers include, but are not limited to, polyanhydrides,poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA),polyg lycolide (PGA), poly(lactide-co-glycolide) (PLGA),poly-3-hydroxybutyrate (PHB) and copolymers thereofpoly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactoneand copolymers thereof, and combinations thereof.

Alternatively, the drug(s) can be incorporated into microparticlesprepared from materials which are insoluble in aqueous solution orslowly soluble in aqueous solution, but are capable of degrading withinthe body by means including enzymatic degradation and/or mechanicalerosion. As used herein, the term “slowly soluble in water” refers tomaterials that are not dissolved in water within a period of 30 minutes.Preferred examples include fats, fatty substances, waxes,, wax-likesubstances and mixtures thereof. Suitable fats and fatty substancesinclude fatty alcohols (such as lauryl, miyristyl stearyl, cetyl orcetostearyl alcohol), fatty acids and derivatives, including but notlimited to fatty acid esters, fatty acid glycerides (mono-, d- andtri-glycerides), and hydrogenated fats. Specific examples include, butare not limited to, hydrogenated vegetable oil hydrogenated cottonseedoil, hydrogenated castor oil, hydrogenated oils available under thetrade name Sterotex®, stearic acid, cocoa butter, and steeryl alcohol.Suitable waxes and wax-like materials include natural or syntheticwaxes, hydrocarbons, and normal waxes. Specific examples of waxesinclude beeswax, glycowax, castor wax , carnauba wax, paraffins andcandelilla wax. As used herein, a wax-like material is defined as anymaterial which is normally solid at room temperature and has a meltingpoint of from about 30 to 300° C.

In some cases, it may be desirable to alter the rate of waterpenetration into the microparticles. To this end, rate-controlling(wicking) agents may be formulated along with the fats or waxes listedabove. Examples of rate-controlling materials include certain starchderivatives (e,g., waxy maltodextrin and drum dried corn starch),cellulose derivatives (e.g., hydroxypropylmethyl-cellulose,hydroxypropylcellulose, methylcellulose, and carboxymethyl-cellulose),alginic acid, lactose and talc. Additionally, a pharmaceuticallyacceptable surfactant (for example, lecithin) may be added to facilitatethe degradation of such microparticles.

Proteins which are water insoluble, such as zein, can also be used asmaterials for the formation of drug containing microparticles.Additionally, proteins, polysaccharides and combinations thereof whichare water soluble can be formulated with drug into microparticles andsubsequently cross-linked to form an insoluble network. For example,cyclodextrins can be complexed with individual drug molecules andsubsequently cross-linked.

Encapsulation or incorporation of drug into carrier materials to producedrug containing microparticles can be achieved through knownpharmaceutical formulation techniques. In the case of formulation infats, waxes or wax-like materials, the carrier material is typicallyheated above its melting temperature and the drug is added to form amixture comprising drug particles suspended in the carrier material,drug dissolved in the carrier material, or a mixture thereof.Microparticles can be subsequently formulated through several methodsincluding, but not limited to, the processes of congealing, extrusion,spray chilling or aqueous dispersion. In a preferred process, wax isheated above its melting temperature, drug is added, and the moltenwax-drug mixture is congealed under constant stirring as the mixturecools. Alternatively, the molten wax-drug mixture can be extruded andspheronized to form pellets or beads. Detailed descriptions of theseprocesses can be found in “Remington—The science and practice ofpharmacy”, 20th Edition, Jennaro et. al., (Phila, Lippencott, Williams,and Wilkens, 2000).

For some carrier materials it may be desirable to use a solventevaporation technique to produce drug containing microparticles. In thiscase drug and carrier material are co-dissolved in a mutual solvent andmicroparticles can subsequently be produced by several techniquesincluding, but not limited to, forming an emulsion in water or otherappropriate media, spray drying or by evaporating off the solvent fromthe bulk solution and milling the resulting material.

In some emhodmients, drug in a particulate form is homogeneouslydispersed in a water-insoluble or slowly water soluble material. Tominimize the size of the drug particles within the composition, the drugpowder itself may be milled to generate fine particles prior toformulation. The process of jet milling, known in the pharmaceuticalart, can be used for this purpose. In some embodiments drug inaparticulate form is homogeneously dispersed in a wax or wax likesubstance by heating the wax or wax like substance above its meltingpoint and adding the drug particles while stirring the mixture. In thiscase a pbannaceutically acceptable; surfactant may be added to themixture to facilitate the dispersion of the drug particles.

The particles can also be coated with one or more modified releasecoatings. Solid esters of fatty acids, which are hydrolyzed by lipases,can be spray coated onto microparticles or drug particles. Zein is anexample of a naturally water-insoluble protein. It can be coated ontodrug containing microparticles or drug particles by spray coating or bywet granulation techniques. In addition to naturally water-insolublematerials, some substrates of digestive enzymes can be treated with,cross-linking procedures, resulting in the formation of non-solublenetworks. Many methods of cross-linking proteins, initiated by bothchemical and physical means, have been reported. One of the most commonmethods to obtain cross-linking is the use of chemical cross-linkingagents. Examples of chemical cross-linking agents include aldehydes(gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, andgenipin. In addition to these cross-linking agents, oxidized and nativesugars have been used to cross-link gelatin (Cortesi, R., et al.,Biomaterials 19 (1998) 1641-1649), Cross-linking can also beaccomplished using enzymatic means; for example, transglutaminase hasbeen approved as a GRAS substance for cross-linking seafood products.Finally, cross-linking can be initiated by physical means such asthermal treatment, UV irradiation and gamma irradiation.

To produce a coating layer of cross-linked protein surrounding drugcontaining microparticles or drug particles, a water soluble protein canbe spray coated onto the microparticles and subsequently cross-linked bythe one of the methods described above.. Alternatively, drug containingmicroparticles can be microencapsulated within protein bycoacervation-phase separation (for example, by the addition of salts)and subsequently cross-linked. Some suitable proteins for this purposeinclude gelatin, albumin, casein, and gluten. Polysaccharides can alsobe cross-linked to form a water-insoluble network. For manypolysaccharides, this can be accomplished by reaction with calcium saltsor multivalent cations which cross-link the main polymer chains. Pectin,alginate, dextran, amylose and guar gum are subject to cross-linking inthe presence of multivalent cations. Complexes between oppositelycharged polysaccharides can also be formed; pectin and chitosan, forexample, can be completed via electrostatic interactions.

Injectable/Implantable Solid Implants

The compositions described, herein can be incorporated intoinjectable/implantable solid implants, such as polymeric implants. Inone embodiment, the compositions are incorporated into a polymer that isa liquid or paste at room temperature, but upon contact with aqueousmedium, such as physiological fluids, exhibits an increase in viscosityto form a semi-solid or solid material. Exemplary polymers include, butare not limited to, hydroxyalkanoic acid polyesters derived from thecopolymerixation of at least one unsaturated hydroxy fatty acidcopolymerized with hydroxyalkanoic acids. The polymer can be melted,mixed with the active substance and cast or injection molded into adevice. Such melt fabrication require polymers having a melting pointthat is below the temperature at which the substance to be delivered andpolymer degrade or become reactive. The device can also be prepared bysolvent casting where the polymer is dissolved in a solvent and the drugdissolved or dispersed in the polymer solution and the solvent is thenevaporated. Solvent processes require that the polymer be soluble inorganic solvents. Another method is compression molding of a mixedpowder of the polymer and the drug or polymer particles loaded with theactive agent.

Alternatively, the compositions can be incorporated into a polymermatrix and molded or compressed into a device that is a solid at roomtemperature. For example, the compositions can be incorporated into abiodegradable polymer, such as polyanhydrides and copolymers thereof,polyhydroalkanoic acids and copolymers thereof, PLA, PGA, and PLGA, andcompressed into solid device, such as disks, or extruded inio a device,such as rods.

Topical Formulations

Suitable dosage forms for topical admintstration include creams,ointments, salves, sprays, gels, lotions, emulsions, and transdermalpatches. The formulation may be formulated for transmucosaLtransepithelial, transendothelial, or transdermal administration.

“Emollients” are an externally applied agent that softens or soothesskin and are generally known in the art and listed, in compendia, suchas the “Handbook of Pharmaceutical Excipients”, 4^(th) Ed.,Pharmaceutical Press, 2003. These Include, without limitation, almondoil, castor oil, teratoma extract, cetostearoyl alcohol, cetyl alcoholeetyl esters wax, cholesterol, cottonseed oil, cyclomehicones ethyleneglycol palmitostearate, glycerin, glycerin monostearate, glycerylmonooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin,light mineral oil, medium-chain triglycerides, mineral oil and lanolinalcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil,starch, stearyl alcohol, sunflower oil, xylitol and combinationsthereof. In one embodiment, the emollients are ethylhexylstearate andethylhexyl palmitate.

“Surfactants” are surface-active agents that lower surface tension andthereby increase the emulsifying, foaming, dispersing, spreading andwetting properties of a product. Suitable non-ionic surfactants includeemulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers,polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters,benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate,poloxamer, povidone and combinations thereof. In one embodiment, thenon-ionic surfactant is stearyl alcohol.

“Emulsifiers” are surface active substances which promote the suspensionof one liquid in another and promote the formation of a stable mixture,or emulsion, of oil and water. Common emulsifiers are: metallic soaps,certain animal and vegetable oils, and various polar compounds. Suitableemulsifiers include acacia, anionic emulsifying wax, calcium stearate,carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol,diethanolamine, ethylene glycol palmitostearate, glycerin monostearate,glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin,hydrous, lanolin, alcohols, lecithin, medium-chain triglycerides,methylcellulose, mineral oil and lanolin alcohols, monobasic sodiumphosphate, monoethanolamine, nonionic emulsifying wax, oleic acid,poloxamer, poloxamers, polyoxyethylene alkyl ethers, polypxyethylenecastor oil derivatives, polyoxyethylene sorbitan fatty acid esters,polyoxyethylene stearates, propylene glycol alginate, self-emulsifyingglyceryl monostearate, sodium, citrate dehydrate, sodium lauryl suflate,sorbitan esters, stearic acid, sunflower oil, tragacanth,triethanolamine, xantban gum and combinations thereof. In oneembodiment, the emulsifier is glycerol stearate.

“Hydrophilic” as used herein refers to substances that have stronglypolar groups that, readily interact with water.

“Lipophilic” refers to compounds having, an affinity for lipids.

“Amphiphilic” refers to a molecule combining hydropbilic and lipophilic(hydrophobic) properties

“Hydrophobic” as used herein, refers to substances that lack an affinityfor water; tending to repel and not absorb water as well as not dissolvein or mix with water.

A “gel” is a colloid in which the dispersed, phase has combined with thecontinuous phase to produce a semisolid material, such as jelly.

An “oil” is a composition containing at least 95% wt of a lipophilicsubstance. Examples of lipophilic substances include but are not limitedto naturally occurring and synthetic oils, fats, fatty acids, lecithins,triglycerides and combinations thereof.

A “continuous phase” refers to the liquid in which solids are suspendedor droplets of another liquid are dispersed, and is sometimes called theexternal phase. This also refers to the fluid phase of a colloid withinwhich solid or fluid particles are distributed. If the continuous phaseis water (or another hydropbilic solvent), water-soluble or hydrophilicdrugs will dissolve in the continuous phase (as opposed to beingdispersed). In a multiphase formulation (e.g., an emulsion), thediscreet phase is suspended, or dispersed in the continuous phase.

As “emulsion” is a composition containing a mixhire of non-misciblecomponents homogenously blended together. In particular emhodiments, thenon-miscible components include a lipophilic component and an aqueouscomponent, An emulsion is a preparation of one liquid distributed insmall globules throughout the body of a second liquid. The dispersedliquid is the discontinuous phase, and the dispersion medium is thecontinuous phase. When oil is the dispersed liquid and an aqueoussolution is the continuous phase, it is known as an oil-in-wateremulsion, whereas when water or aqueous solution is the dispersed phaseand oil or oleaginous substance is the continuous phase,it is known as awater-in-oil emulsion. Either or both of the oil phase and the aqueousphase may contain one or more surfactants, emulsifiers, emulsionstabilizers, buffers, and other excipients. Preferred excipients includesurfactants, especially non-ionic surfactants; emulsifying agents,especially emulsifying waxes; and liquid, non-volatile non-aqueousmaterials, particularly glycols such as propylene glycol. The oil phasemay contain other oily pharmaceuticaily approved excipients. Forexample, materials such as hydroxylated castor oil or sesame oil may beused in the oil phase as surfactants or emulsifiers.

An emulsion is a preparation of one liquid distributed in small globulesthroughout the body of a second liquid. The dispersed liquid is thediscontinuous phase, and the dispersion medium is the continuous phase.When oil is the dispersed liquid and an aqueous solution is thecontinuous phase, it is known as an oil-in-water emulsion, whereas when,water or aqueous solution is the dispersed phase and oil or oleaginoussubstance is the continuous phase, it is known as a water-in-oilemulsion. The oil phase may consist at least in part of a propellant,such as an HFA prepellent. Either or both of the oil phase and theaqueous phase may contain one or more surfactants, emulsifiers, emulsionstabilizers, buffers, and other excipients. Preferred excipients includesurfactants, especially non-ionic surfactants; emulsifying agents,especially emulsifying waxes; and liquid non-volatile non-aqueousmaterials, particularly glycols such as propylene glycol. The oil phasemay contain other oily pharmaceutically approved excipients. Forexample, materials such as hydroxylated castor oil or sesame oil may beused in the oil phase as surfactants or emulsifiers.

A sub-set of emulsions are the self-emulsifying systems. These drugdelivery systems are typically capsules (hard shell or soft shelf)comprised of the drug dispersed or dissolved in a mixture ofsurfactant(s) and lipophilic liquids such as oils or other waterimmiscible liquids. When the capsule is exposed to an aqueousenvironment and the outer gelatin shell dissolves, contact between theaqueous medium and the capsule contents instantly generates very smallemulsion droplets. These typically are in the size range of micelles ornanoparticles. No mixing force is required to generate the emulsion asis typically the case in emulsion formulation processes.

A “lotion” is a low- to medium-viscosity liquid formulation. A lotioncan contain finely powdered substances that are in soluble in thedispersion mediumthrough the use of suspending agents and dispersingagents. Alternatively, lotions can have as the dispersed phase liquidsubstances that are immiscible with the vehicle and are usuallydispersed by means of emulsifying agents or other suitable stabilizers.In one embodiment, the lotion is in the form of an emulsion having aviscosity of between 100 and 1000 centistokes. The fluidity of lotionspermits rapid and uniform application over a wide surface area. Lotionsare typically intended to dry on the skin leaving a thin coat of theirmedicinal components on the skin's surface.

A “cream” is a viscous liquid or semi-solid emulsion of either the“oil-in-water” or “water-in-oil type”. Creams may contain emulsifyingagents and/or other stabilizing agents. In one embodiment, theformulation is in the form of a cream having a viscosity of greater than1000 centistokes, typically in the range of 20,000-50,000 centistokes.Creams are often time preferred over ointments as they are generallyeasier to spread and easier to remove.

The difference between a cream and a lotion is the viscosity, which isdependent on the amount/use of various oils and the percentage of waterused to prepare the formulations. Creams are typically thicker thanlotions, may have various uses and often one uses more variedoils/butters, depending upon the desired effect upon the skin. In acream formulation, the water-base percentage is about 60-75 % and theoil-base is about 20-30 % of the total, with the other percentages beingthe emuisifler agent, preservatives and additives for a total of 100%.

An “ointment” is a semisolid preparation containing an ointment base andoptionally one or more active agents. Examples of suitable ointmentbases include hydrocarbon bases (e.g., petrolatum, white petrolatum,yellow ointment, and mineral oil); absorption bases (hydrophllicpetrolatum, anhydrous lanolin, lanolin, and and cream); water-removablebases (e.g., hydrophillic ointment), and water-soluble bases (e.g.,polyethylene glycol ointments). Pastes typically differ from ointmentsin that they contain a larger percentage of solids. Pastes are typicallymore absorptive and less greasy that ointments prepared with the samecomponents.

A “gel” is a semisolid system containing dispersions of small or largemolecules in a liquid, vehicle that is rendered semisolid by the actionof a thickening agent or polymeric material dissolved or suspended inthe liquid vehicle. The liquid may include a lipophilic component, anaqueous component or both. Some emulsions may be gels or otherwiseinclude a gel component. Some gels, however, are not emulsions becausethey do not contain a homogenized blend of immiscible components.Suitable gelling agents include, but are not limited to, modifiedcelluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose;Carbopol homopolymers and copolymers; and combinations thereof. Suitablesolvents in the liquid vehicle include, but are not limited to, diglycolmonoethyl ether; alklene glycols, such as propylene glycol; dimethylisosorbide; alcohols, such as isopropyl alcohol and ethanol. Thesolvents are typically selected for their ability to dissolve the drug.Other additives, which improve the skin feel and/or emolliency of theformulation, may also be incorporated. Examples of such additivesinclude, but are not limited, isopropyl myristate, ethyl acetate,C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone,capric/caprylic triglycerides, and combinations thereof.

Foams consist of an emulsion in combination with a gaseous propellant.The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs).Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3- heptafluoropropane (HFA 227), but mixtures andadmixtures of these and other HFAs that are currently approved or maybecome approved for medical use are suitable. The propellants preferablyare not hydrocarbon propellant gases which can produce flammable orexplosive vapors during spraying. Furthermore, the compositionspreferably contain no volatile alcohols, which can produce flammable orexplosive vapors daring use.

Buffers are used to control pH of a composition. Preferably, the buffersbuffer the composition from a pH of about 4 to a pH of about 7.5, morepreferably from a pH of about 4 to a pH of about 7, and most preferablybom a pH of about 5 to a pH of about 7. In a preferred embodiment, thebuffer is triethanolamine.

Preservatives can be used to prevent the growth of fungi andmicroorganisms. Suitable antifungal and antimicrobial agents include,but are not limited to, benzoic acid, butylparaben, ethyl paraben,methyl paraben, propylparaben, sodium, benzoate, sodium propionate,benzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,and thimerosal.

The term aerosol as used herein refers to any preparation of a fine mistof particles, which can be in solution or a suspension, whether or notit is produced using a propellant. Aerosols can be produced usingstandard techniques, such as ultrasonication or high pressure treatment.See, for example, Adjel, A. and Garren, J. Pharm. Res., 7: 565-569(1990); and Zanen, P. and Lamm, J,-W. J, Int. J. Pharm., 114; 111-415(1995). Preferably, the aqueous solutions is water, physiologicallyacceptable aqueous solutions containing salts and/or buffers, such asphosphate buffered saline (PBS), or any other aqueous solutionacceptable for administration to a animal or human. Such solutions arewell knows to a person skilled in the art and include, but are notlimited to, distilled water, de-ionized water, pore or ultrapure water,saline, phosphate-buffered saline (PBS). Other suitable aqueous vehiclesinclude, but are not limited to. Ringer's solution and isotonic sodiumchloride. Aqueous suspensions may include suspending agents such ascellulose derivatives, sodium alginate, polyvinylpyrrolidone and gumtragacanth, and a wetting agent such as lecithin. Suitable preservativesfor aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

In another ernhodiment, solvents that are low toxicity organic (i.e.nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethylacetate, tetrahydofuran, ethyl ether, and propanol may be used for theformulations. The solvent is selected based on its ability to readilyaerosolize the formulation. The solvent should not detrimentally reactwith the compositions. An appropriate solvent should be used thatdissolves the compositions or forms a suspension of the compositions.The solvent should be sufficiently volatile to enable formation of anaerosol of the solution or suspension. Additional solvents oraerosolizing agents, such as freons, can be added as desired to increasethe volatility of the solution or suspension.

III. Methods of Administration

The composition can be used for any of the methods for administeringlocal anesthetics known to one of ordinary skill in the art. Thecomposition can be formulated for topical anesthesia, infiltrationanesthesia, filed block anesthesia, nerve block anesthesia, intravenousregional anesthesia, spinal, anesthesia and epidural anesthesia.

The anesthetic will typically be provided as a solution or as alyophilized powder or in crystalline form which is reconstituted at thetime of use with a diluent typically packaged with the anesthetic.Either may include the CPE. For a site I sodium channel blocker such asTTX/STX, the CPE will be present in excess due to the extremely smallamount of local anesthetic required. The anesthetic will typically berelatively dilute for safety reasons, as described in the examples. Thesolution is typically slightly acidic for stability reasons, but woulddepend on the CPE. The pH important to the extent that most site Iblockers are stored (if a liquid) in acidic pH (typically less than5.5).

IV. EXAMPLES

The present Invention will be further understood by reference to thefollowing non-limiting examples.

Example 1 Comparison of Site I Sodium Channel Blocker or LocalAnesthetic with and without Surfactant CFE

Materials and Methods

Animd Care. Young adult male Sprague-Bawley rats (350-420 g) wereobtained from Charles River Laboratories (Wilmington, Mass.) and housedin groups of two per cage on a 6 a.m. to 6 p.m, light/dark cycle. Allanimals were cared for in accordance with protocols approvedinstitutionally and nationally.

Chemical Enhancers & Solution Preparation. Representative enhancers fromthree different classes of surfactants were obtained from Sigma (St.Louis, Mo.): sodium lauryl sulfate (SLS) and sodium octyl sulfate (SOS),.anionic surfactants; dodecyltriethylammonium bromide (DDAB) andoctyltriethylammonium bromide (OTAB), cationic surfactants; and Tween®20 and Tween® 80, nonionic surfactants (Table 1).

Tetrodotoxin (TTX) and bupivacaine (Sigma) solutions were prepared insaline individually and in combination with each enhancer the nightbefore scheduled injections. TTX and bupivacaine concentrations werechosen to be near the bottom of their respective dose-response craves(Kohane, et al., Anesthesiology, 89:1199-1208 (1998); Kohane, et al.,Reg Anesth Pain Med, 26:239-45 (2001); enhancer concentrations wereinitially chosen to be approximately 50% of those used successfully intransdermal applications, followed by lower and higher concentrations asneeded to obtain a dose-response curve.

Sciatic Blockade Technique. Animals were cared for in compliance withprotocols approved by the Massachusetts Institute of Technology (MIT)Committee on Animal Care, in conformity with the NIH guidelines for thecare and use of laboratory animals (NIH publication #5-23, revised1985). Rats were anesthetized using isoflurane in oxygen. A 23-guageneedle was introduced posteromedial to the greater trochanter, and 0.3mL of the drug +/− enhancer solution was injected upon contacting bone.To generate the dose-response curves for the CPEs, four animals wereinjected with, each CPE at 5 different concentrations (each alone andwith 30 μM TTX). Larger sample sizes were obtained at the followingconcentrations that were important for further experiments: 11 mM SOS, 2mM SLS, 9 mM SLS, 17 mM SLS, 35 mM SLS, 46 mM Tween 80 (n=8); 4 mM SLS(n=16); and 23 mM Tween 80 (n=22). For CPEs injected with 1.39 mMbuph/aealne, n=4 for each CPE-bupivacaine combination, A total of 12animals were injected with 139 mM bupivacaine alone (no CPE).

Assessment of Nerve Blockade. In all experiments, the experimenter wasblinded as to what treatment any given rat had received. Presence andextent of nerve blockade was investigated as previously described(Kohane, et al., Anesthesiology, 89:1199-1208 (1998); Padera, et al.,Muscle Nerve, 34:747-53 (2006); Kohane, et al;, Anesthesiology,89:119-31 (1998); Masters, et. al;, Anesthesiology, 79(2):340-346(1993)). Erratum in: Anesthesiology, 79(5): 1160 (1993)). Briefly,thermal nociception of each leg was assessed, with the right(uninjected) leg serving as an untreated control.

Thermal nociception was assessed by a modified hotplate test. Hind pawswere exposed in sequence (left then right) to a 56° C. hot plate (Model39D Hot Plate Analgesia Meter, IITC Inc., Woodland Hills, Calif.). Thetime (latency) until paw withdrawal was measured with a stopwatch. Ifthe animal did not remove its paw from the hot plate within 12 seconds,it was removed by the experimenter to avoid injury to the animal or thedevelopment of hyperalgesia. The duration of thermal nociceptive blockwas calculated as the time required for thermal latency to return to avalue of 7 seconds from a higher value. Seven seconds is the midpointbetween a baseline thermal latency of approximately 2 seconds in adultrats, and a maximal latency of 12 seconds. Latencies>7 sec wereconsidered to be effective blocks.

Kohane, et al., Anesthesiology, 89:1199-1208 (1998); Kohane, et al.,Anesthesiology 90:524-34 (1999 demonstrated >99% successful, blocks with0.1-03 mL of 0.25%-0.5% (8.7-17 mM) bupivacaine, indicating thatdifferences in block duration reflected actual pharmacologicaldifferences rather than operator error.

Tissue Harvesting and Histology. Animals were euthanized with carbondioxide, and the sciatic nerves and adjacent tissues were harvested forhistology. Tissues were fixed in Accustain (formalin-free fixative)company, city, state, embedded in paraffin, sectioned, and stained withhematoxylin and eosin by the Department of Comparative Medicine at MIT(foe for service), using standard techniques.

Cell Culture. C 2C12 , a mouse myoblast ceil line (American Type CultureCollection, ATGC, CRL-1772, Manassas, Va.) was cultured to proliferatein Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% FetalBovine Serum and 1% Penicillin Streptomycin (Pen Strep). All cellculture supplies were purchased from Invitrogen (Carlsbad, Calif.)unless otherwise noted. Cells were plated in 24-well tissue cultureplates whh 50,000 cells/mL/well in DMEM supplemented with 2% Horse Serumand 1% Pen Strep, and left, to differentiate into myotubules for 10-14days. During differentiation, media was exchanged every 2 to 3 days.After one week of myotube differentiation, 100 μL of the 10x enhancer+/− TTX containing media was added to 900 μL of fresh media; 100 μL ofPBS was added. In control wells. The enhancer solution was prepared bydissolving the enhancer in PBS and stirring overnight. The solution wasfiltered, aseptically using a 0.2 μm syringe filter. The TTX, or PBS forgroups not containing drag, was added aseptically to the enhancersolution. At 2, 8, 24, or 96 hoors the plates were assayed as describedbelow. Cells were maintained at 37° C. in 5% CO₂ balance air.

Assessing viability. Cell viability was assessed using a colorimetricassay (MTT kit, Promega G4100 Madison, Wis.) in which a yellowtetrazollum salt (MTT) is metabolized in live cells to form insolublepurple formazan crystals. The purple crystals are solobilized by theaddition of a detergent, and the color is then be quantifiedspectrophotometrically. At each time point 150 μL of MTT reagent wasadded to the cells. Following a four hour incubation at 37° C. 1 mL ofsolubilization solution (detergent) was added. The absorbance was readat 570 nm using the SpeetraMax 384 Plus fluorometer (Molecular Devices,Sunnyvale, Calif.) after overnight incubation. Cells were also monitoredvisually to confirm the results of the MTT assay. Each plate containedmedia only wells whose absorbance was subtracted from the rest of theplate as noise. All groups were then normalized to blank media.

Statistical Analysis. In vivo neurobehavioral data were not normallydistributed, and are therefore presented as medians with 25^(th) and75^(th) percentiles and compared by Mann-Whitney U-test. MIT assayresults are described parametrically with means +/− standard deviationsand compared by t-tests and analysis of variance (ANOVA). Statisticalsignificance, for both parametric and nonparametric tests, was definedas P<0.05.

Results

Effect of Enhancers on Nerve Blockade with TTX.

Injection of 0.3 mL of 30 μM TTX caused sensory blockade in 29% ofanimals tested (n=24). The median duration of block was 0 minutes, with25^(th) and 75^(th) percentiles of 0 minutes and 47 minutes,respectively. The selected concentration was based on Kohane, et al.,Anesthesiology, 89:119-31 (1998) and chosen for further studies asimprovement of nerve blockade could easily be detected. Dose-responsecurves were obtained for the duration, of block from 30 μM TTX withvarying concentrations of SLS and SOS (anionic surfactants), DDAB andOTAB (cationic surfactants), and Tween® 20 and Tween® 80 (nonionicsurfactants) individually (FIG. 1). The group that received DDAB wasalso injected with 30 μM. TTX in 32 mM of the enhancer, but none ofthose rats' nerve blocks resolved (returned to normal).

In most cases, the enhancers demonstrated a concentration-dependentincrease in block duration before reaching a plateau. This maximalduration of block was used as a measure of efficacy for all CPEs exceptfor DDAB, where the longest duration of block from, which the animalsrecovered was used. There was a range of maximum durations of block dueto the CPEs, the greatest being seen with DDAB (median maximum durationof block=606 minutes; FIG. 1, Table 2).

TABLE 2 Effect of chemical penetration enhancers (CPEs) on the durationof nerve blockade from TTX. Maximum TTX 30 μM Block Duration EC_(MBD)EC_(100 min) EC_(50eff) with: (MBD, minutes) (mM) (mM) (mM) — 0 (0-47) — — — SOS 521 (361-669) 86 22 32 SLS 240 (255-277) 35 3 4 OTAB 353(327-361) 119 31 30 DDAB 606 (452-788) 8 1 6 TW 20 163 (129-206) 49 4 3TW 80 120 (111-143) 81 40 34

CPEs markedly prolonged the maximum block duration (MBD) from 30 μm TTX.The EC_(MBD) is the CPE concentration that caused the maximumprolongation of block front 30 μm TTX, The EC_(100min) is theinterpolated concentrationof CPE that increased the block duration of 30μm TTX to 100 min. The EC_(50eff) is the concentration of a given CPEthat caused a half-maximal increase in duration of block (half-MBD) of30 μm TTX. Block durations are medians with 25^(th) and 75^(th)percentiles in parentheses; n=4 for all CPE concentrations, except thosespecified in Materials and Methods (where n=8-22).

As shown in Table 3 below, all the CPEs increased the percentage ofanimals that developed effective nerve block from 29.2% with TTX aloneto 88-100%.

TABLE 3 Concentrations of chemical penetration enhancers (CPEs)producing maximal frequency of nerve blockade with 30 μM TTX. Highest %CPE TTX 30 μm Animals with concentration with: Effective Block (mM) — 29— SOS 100 32 SLS 88 17 OTAB 100 40 DDAB 100 6 Tween 20 100 4 Tween 80100 81

CPEs increased the percentage of animals developing effective block from30 μm TTX. Blocks were considered effective if latency was >7 seconds atany point. The CPE concentration listed was the lowest needed to achievethe highest percentage of animals with block; n=4 for all conditionsexcept TTX alone (n=24) and TTX+SLS (n=8).

At the concentrations needed, to cause this maximum percentage of block,all CPEs significantly improved TTX block duration (for SOS, SLS, OTAB,DDAB, Tween® 20, and Tween® 80, p<0.001 compared to the duration ofblock from TTXalone), in general, the maximum prolongation of TTX blockby the cationic surfactants (OTAB, DDAB) was statistically significantlygreater than, prolongation by the other CPEs. The maximum prolongationof TTX block by the nonionic surfactants (Tween® 20, Tween® 80) wasgenerally less than that by the others (p<0.05 by Mann-Whitney U-test).

As a measure of the potency of the block-prolonging effect of the CPEs,it was determined by interpolation that the concentration of eachenhancer required to achieve a duration of block of 100 minutes (theEC_(100min>) Table 2). Tween® 20, SLS, and DDAB were more potent (lowerEC_(100min)) than Tween® 80, SOS, and OTAB (FIG. 1; Table 2). Within theclasses of enhancers where the members differed only in the length ofthe carbon chain (SOS-SLS; OTAB-DDAB), those with the eight-carbon chainwere less potent than those with the twelve-carbon chain, (In the caseof the Tween® compounds, that relationship was reversed, but there areother significant differences in the Tween® structures.) Each enhancerwas also injected alone, without TTX, to confirm that the increasedduration of block was not due to analgesic or toxic effects of theenhancers themselves; with one exception (32 mM DDAB), the CPE alone didnot cause nerve blockade.

Effect of Enhancers on Nerve Blockade with Bupivacaine.

139 mM (0.04%) bupivacine had performance characteristics comparable to30 μm TTX with respect to percentage of animals blocked and medianduration of block (Table 4) by producing a dose-response curve ofsciatic nerve blocks with six bupivacaine concentrations spanning therange 0.69 mM-6.9 mM, (n=4-12 rats per concentration). 1.39 mMbupivacaine was tested with each of the CPEs at a concentration thatcreated a half-maximal increase in the duration of block of TTX, theEC_(50eff) (Table 2). None of the 6 enhancers tested in theseexperiments resulted in a statistically significant change in theduration of bnpivaeaine nerve block as shows in Table 4.

TABLE 4 Effect of chemical penetration enhancers (CPEs) on frequency ofnerve blockade from bupivacaine. Bupivacaine 1.4 mM Duration of %Animals with the Sensory Block with Effective EC_(50eff of): (minutes)Block —  0 (0.34)  42 (5/12) SOS 0 (0-8) 25 (1/4) SLS 22 (0-60) 50 (2/4)OTAB 0 (0-0)  0 (0/4) DDAB 0 (0-0)  0 (0/4) Tween 20  0 (0-19) 25 (1/4)Tween 80 0 (0-0)  0 (0/4)

Duration and frequency of nerve block from bupivacaine with and withouteach of the GPEs at the EC_(50eff) from Table 2a. Durations of effectiveblock (DEB) are expressed as medians with 25^(th) and 75^(th)percentiles in parentheses. For bupivacaine alone, n=12; forbupivaeaine+CPE, n=4. In vitro toxicity.

C2C12 myotuhe cultures were exposed to each enhancer at its EC_(50eff)with and without TTX and assayed for viability after 2 hours (FIG. 2).The most toxic enhancers were DDAB and SOS, followed hy SLS, OTAB.Tween® 80, and Tween° 20, in order of decreasing toxicity. C2C12viability decreased with increased duration of exposure to all CPEsexcept Tween® 20, which remained at untreated-control levels after an8-hour exposure (data not shown). Addition of TTX to the ceil culturemedium did not impact cell survival when given alone or in the presenceof enhancers.

In vivo toxicity.

The sciatic nerves and surrounding muscle of rats injected with theEC_(50eff) of each enhancer (i.e., the same concentration used invitro), with and without TTX, were examined for evidence of inflammationand tissue injury four days after injection. Four animals were injectedin each group.

Animals injected with the EC_(50eff) of SLS, OTAB, Tween® 20 and Tween®80 showed no significant muscle or nerve injury, although some samplesin all groups showed mild inflammation with macrophages and lymphocytesaround the muscle and nerve, without evidence of infiltration, fibrosis,or atrophy within the muscle or nerve. Because Tween® 20 at itsEC_(50eff) showed no evidence of toxicity in vitro or in vivo,additional concentrations were tested to determine the highest sub-toxicconcentration. Tweenn® 20 at 24.4 and 81.4 mM showed progressivelyworsening (mild to moderate) muscle atrophy and inflammation, similar intype but not severity to that seen with the DDAB EC_(50eff). Note that81.4 nM is more than twenty times the EC_(50eff) Tween® 20. Samples fromanimals injected with DDAB consistently showed moderate to severeinfiltration of macrophages and lymphocytes, atrophy and degeneration ofmuscle fibers, and fibrosis of the tissue. An additional two animalswere injected with 3% (97.3 mM) DDAB (the concentration at which animalsdeveloped irreversible nerve block). These showed deep and severe tissuedamage, including ischemic necrosis, accompanied by severe and extensiveinflammation.

Animals injected with the EC_(50eff) the enhancers together with 30 μmTTX showed the same histological results as those without TTX. Again,some of the samples exposed to DDAB showed severe lymphocyticinflammatory infiltration of muscle with degenerative changes,regenerative changes, and fibrosis. These samples also showed a mildlymphocytic infiltrate of nerve and focal fat necrosis.

Indicators of nerve fiber injury, including fibrosis and myelin ovoids,were not seen in any samples, but subtle degrees of damage to myelinatednerve fibers cannot be accurately assessed using paraffin-embedded,hematoxylin-eosin-stained sections.

Discussion

Surfactant CPBs caused a concentration-dependent increase in TTX-mdueednerve block, but, at the concentrations tested here, did not enhanceblock from bupivacaine. This difference is due to TTX being extremelyhydrophillc, having an obligate charge, while bupivacaine, like allamino-ester and amino-amide local anesthetics, can be conditionallyhydrophobic due to its aromatic moiety and tertiary amine. There is apH-dependent equilibrium between the cationic protonated form ofbupivacaine that is water soluble and the neutral form that is solublein organic solvents (i.e. is hydrophobic), and therefore partitionsrelatively easily into cell membranes and other biological barriers. Therelatively pronounced improvement in block from TTX with CPEs relates toits lack of hydropbobicity, whereas bupivaecaine does not benefitbecause its structure already permits easy crossing of biologicalbarriers. High (millimolar) concentrations of adrenergic antagonists,far in excess of the range in which they are active on adrenergicreceptors, greatly prolong the duration of block by TTX (Kohane, et al.,Reg Anesth Pain Med, 26:239-45 (2001)). The results presented heresupport the view that the prolongation of nerve block by thosepolycyclic compounds was due to flux enhancement (i.e. those compoundswere acting as CPEs).

All the CPEs examined resulted in prolongation of TTX block. Thoughthere was a considerable range in the magnitude of enhancement, noindividual CPE or class of CPE (anionic, cationic, or nonionicsurfactant) clearly performed belter than all the others. The nonionicagents' block prolongations, though significant, were shorter than thoseof the other CPEs. This is consistent with the effects of surfactants onpermeant flux across the stratum corneum and epidermis of the skin(Kushla, et al., J Pharm Sci, 82:1118-1122 (1993). The various CPEsresulted in a variety of patterns of block prolongation with respect tothe magnitude of the increase in the maximum duration of block, or theimprovement (reduction) in the EC_(100min). It is important to becareful in using the EC_(100min) to make comparative statementsregarding potency, since the shapes of the dose-response curves tor eachCPE are not always similar. In general, the magnitude of the maximalimprovement in duration of block (the maximum block duration, MBD) didnot correlate well with the potency (EC_(100min), FIG. 3). There alsowas no consistent pattern in the effect of hydrophobic chain length onduration of block.

CPEs varied widely in the cytoxicity of their EC_(50eff). In general,the agents that produced the longest maximal block durations were moretoxic in cell culture (FIG. 4, R²=0.66). There was no correlationbetween the EC_(100min) and toxicity (R²=0.11). With the cationicsurfactants, toxicity increased with molecular weight and carbon-chainlength, while it decreased with the same parameters in anionicsurfactants.

In assessing the balance between maximum block duration and cytotoxicity(shown in Table 5 below), Tween® 20 would appear to have the mostfavorable relevant ratios.

TABLE 5 Relationship between maximum block duration (MBD) andEC_(100 min), and in vitro viability. TTX 30 μm MBD % Survival with: %Survival (100 − % Survival) EC_(100 min) SOS  5 ± 3 5.5 0.2 SLS  17 ± 112.9 5.7 OTAB 53 ± 2 7.5 1.7 DDAB   5 ± 0.2 6.4 5.0 Tween 20 94 ± 3 27 24Tween 80 80 ± 8 6.0 2.0

The maximum block duration (Table 2), EC_(100min) (Table 1), and invitro survival, data (determined from C2C12 MIT assay). The values formaximal block duration and EC_(100min) are from Table 2; those for cellsurvival are derived from FIG. 2. Cell survival data are meanpercentages with standard deviations. For the two ratios in the columnson the right, a high value is favorable (good ratio of performance totoxicity).

The in vivo data showed that all compounds, with the notable exceptionof DDAB, caused little or no tissue injury when delivered at the sameconcentrations as used in vitro (the EC_(50eff), which had causedapproximately half-maximal increase in duration of block from TTX). Thisdiscrepancy may be explained by differences between cultured cell linesand in vivo tissue, but it is also possible that the local concentrationof the CPEs dissipates rapidly after injection in vivo. Tetrodotoxinitself caused little or no toxicity, with or without enhancers, afinding consistent with Kohane, et al., Anesthesiology 89:1199-1208(1998). The in vivo results indicate that enhancer toxicity can beminimal or non-existent within a concentration range that results insignificant block duration, and that the most efficacious compoundscould be used rather than those with the best toxicity profile thorn invitro studies. There was little or no evidence of direct nerve injury inall the CPEs investigated, including concentrations of DDAB thatresulted in long-term loss of nerve function.

Myotoxicity and neurotoxicity are well-known concomitants ofconventional ammo-ester and amino-amide local anesthetics, but not oftetrodotoxin (Padera, et al., Muscle Nerve, 34:747-53 (2006); Benoit, etal., Toxicol Appl. Pharmacol., 52:187-198 (1980); Sakura, et al., AnesthAnalg., 81:338-346 (1995). TTX's principal disadvantage is systemictoxicity, which is dose-limiting. In these experiements, CPEsdramatically increased the median duration of block from a very lowconcentration of TTX (e.g. from 0 to 353 min by use of OTAB). Thesedurations of block far exceed those that could be achieved even bytoxic, near-lethal concentrations of TTX in the absence ofvasoconstrictors. For example, 50 μm TTX applied in the same mannerwithout CPEs resulted in an average duration of block of approximately150 minutes, but with a 20% mortality rate (Kohane, et al.,Anesthesiology, 89:119-31 (1998). It follows that the use of CPEs wouldresult in a marked improvement in the therapeutic index of TTX (theratio of the effective to the lethal dose).

Flux enhancing agents caused a marked increase in nerve blockadeduration from hydrophilic TTXs but did not improve block duration fromamphiphilic bupivacaine. The prolongation, of TTX block was provided bydifferent types of surfactants. Although there was considerablecytotoxicity from some CPEs in vitro, histology from in vivo experimentsshowed little or no damage in muscle and nerve, except with DDAB.

Example 2 Effect Combining Site I Sodium Channel Blocker with LocalAnesthetic Materials and Methods

Animal Care

Young adult, male Sprague-Dawley rats (350-420 g) were obtained fromCharles River Laboratories (Wilmington, Mass.) and housed its groups oftwo per cage on a 6 a.m to 6 p.m. light/dark cycle. All animals werecared for in accordance with protocols approved institutionally andnationally.

Chemical Enhancers & Solution Preparation

QX-314 and QX-222 (Sigma) solutions were prepared in saline individuallythe night before scheduled injections.

Sciatic Blockade Technique

Animals were cared for in compliance with protocols approved by theMassachusetts Institute of Technology (MIT) Committee On Animal Care, inconformity with the NIH guidelines for the care and use of laboratoryanimals (NIH publication #85-23, revised 1985). Rats were anesthetizedusing isoflurane in oxygen. A 25-guage needle was Introducedposteromedial to the greater trochanter, and 300 μL, injected uponcontacting bone.

Assessment Nerve Blockade

In all experiments, the experimenter was blinded as to what treatmentany given rat had received. Presence and extent of nerve blockade wasinvestigated as previously described (Kohane, et al., Anesthesiology,89:1199-1208 (1998); Padera, et al., Muscle Nerve, 34:747-53 (2006);Kohane, et al., Anesthesiology, 89; 119-31 (1998); Masters, et al.,Anesthesiology, 79(2):340-346 (1993)). Briefly, thermal nociception ofeach leg was assessed, with the right (uninfected) leg serving as anuntreated control.

Thermal nociception was assessed by a modified hotplate test. Hind pawswere exposed in sequence (left then right) to a 56° C hotplate (Model39D Hot Plate Analgesia Meter, IITC Inc., Woodland Hills, Calif.). Thetime (latency) until paw withdrawal was measured with a stopwatch. Ifthe animal did not remove its paw from, the hot plate within 12seconds,, it was removed by the experimenter to avoid injury to theanimal or the development of hyperalgesia. The duration of thermalnociceptive Mock was calculated as the time required for thermal latencyto return to a value of 7 seconds from a higher value. Seven seconds isthe midpoint between a baseline thermal latency of approximately 2seconds in adult rats, and a maximal latency of 12 seconds. Latenciesgreater than 7 sec were considered to be effective blocks.

Extensor Postural Thrust (EPT)

The rat was held with its posterior placed at a digital balance on whichit could bear weight with one hindpaw at a time. The maximum weight thatthe rat could hear without its ankle touching the balance was measured.

Anesthetic

30 μM, 40 μM, or 50 μM TTX, 25 and 70 mM QX-314 and 70 and 100 mM QX-222were tested alone or in combination for thermal nociceptor and motorblockade.

Results

The results are shown, in FIGS. 5A and B. The combination of QX-314 orQX-222 with TTX increased blockade significantly more than the merecumulative value of the anesthetics alone.

We claim:
 1. A method for enhancing nerve blockade without significant cytotoxicity comprising administering a site I sodium channel local anesthetic in combination with an effective amount of chemical permeation enhancer selected from the group consisting of surfactants, terpenes, amino amides, amino esters, azide-like compounds and alcohols, to increase blockade more than the additive effect of the local anesthetic or chemical, permeation enhancer alone.
 2. The method of claim 1 wherein the local anesthetic is selected from the group consisting of tetrodotoxin (TTX), saxitoxin (STX), decarbamoyl saxitoxin, neosaxitoxin, and the gonyanloxins.
 3. The method of claim 1 wherein the chemical penetration enhancer is selected from the group consisting of anionic, cationic, and nonionic surfactants.
 4. The method of claim 1 further comprising, administering a vasoconstrictor.
 5. The method of claim 1 further comprising a charged local anesthetic selected from the group consisting of amino-amide or amino-ester local anesthetics or derivatives thereof, at least partly amphophilic local anesthetics, local anesthetics that act not on the surface of the cell, and at least partly charged local anesthetics.
 6. The method of claim 1 comprising providing a kit comprising a vial having the local anesthetic therein and a second vial containing a diluent therein.
 7. The method of claim 1 comprising administering the anesthetic as a bolus dosage unit.
 8. The method of claim 1 comprising administering the anesthetic in a continuous or sustained release formulation.
 9. The method of claim 1 comprising administering the anesthetic in a topical or aerosol formulation.
 10. An anesthetic composition for enhancing nerve blockade without significant cytotoxicity comprising a site I sodium channel local anesthetic in combination with an effective amount of chemical permeation enhancer selected from the group consisting of surfactants, terpenes, amino amides, amino esters, azide-like compounds and alcohols, to increase blockade more than the additive effect of the local anesthetic or chemical permeation enhancer alone.
 11. The composition of claim 10 wherein the local anesthetic is selected from the group consisting of tetrodotoxin (TTX), saxitoxin (STX), decarbamoyl saxitoxin, neosaxitoxin, and the gonyautoxins.
 12. The composition of claim 10 wherein the chemical penetration enhancer is selected from the group consisting of anionic, cationic, and nonionic surfactants.
 13. The composition of claim 10 further comprising a vasoconstrictor.
 14. The composition of claim 10 further comprising a charged local anesthetic selected from the group consisting of amino-amlde or amino-ester local anesthetics or derivatives thereof, at least partly amphiphilic local anesthetics, local anesthetics that act not on the surface of the cell, and at least partly charged local anesthetics.
 15. The composition of claim 10 in a kit comprising a vial comprising the local anesthetic therein and a second vial comprising a diluent therein.
 16. The composition of claim 10 comprising the anesthetic as a bolus dosage unit.
 17. The composition of claim 10 comprising the anesthetic in a continuous or sustained release formulation.
 18. The composition of claim 10 comprising the anesthetic in a topical or aerosol formulation.
 19. A method for providing local anesthesia comprising providing an effective amount of a site I sodium channel, blocker in combination with a local anesthetic selected from the group consisting of a charged local anesthetic selected from the group consisting of amino-amide or amino-ester local anesthetics or derivatives thereof, at least partly amphiphilic local anesthetics, local anesthetics that act not on the surface of the cell, and at least partly charged local anesthetics.
 20. The method of claim 19 wherein the local anesthetic is a lidocaine or lidocaine derivative.
 21. A composition for providing local anesthesia comprising an effective amount of a sited sodium channel blocker in combination with a local anesthetic selected from the group consisting of a charged local anesthetic selected from the group consisting of amino-amide or amino-ester local anesthetics or derivatives thereof, at least partly amphophilic local anesthetics, local anesthetics that act not on the surface of the cell and at least partly charged local anesthetics.
 22. The composition of claim 21 wherein the local anesthetic is a lidocaine or lidocaine derivative. 